Glucose levels in the different treatment groups.glucose absorption. This would also mean, that in case of a welldesigned mulberry preparation for anti-diabetic purposes the high chlorogenic acid and rutin content should be accompanied by low levels of certain undesired flavonoid(s) ?future research is needed to clarify whether such criteria are necessary or not.ConclusionsOur results can briefly be summarized as follows. 1. A significant, dose-dependent anti-diabetic activity was found for the 70 aqueous ethanolic extract of Morus alba leaves on our in vivo model of type II. diabetic rats. 2. An analitical Benzocaine site method was developed for the rapid, selective determination of three, potentially active, major constituents (chlorogenic acid, rutin and isoquercitrin) of the extract by HPLC-DAD. 3. Contribution of the three major constituents to the overall activity was investigated, and a dose related activity wasGroups Control Glibenclamide MA (250 mg/kg) MA (750 mg/kg) 1 (9 mg/kg) 1 (27 mg/kg) 2 (5 mg/kg) 2 (15 mg/kg) 3 (3 mg/kg) 3 (9 mg/kg)Day 0 6.3260.41 5.5260.39 7.3160.80 5.6060.27 6.0860.48 6.1160.41 6.7961.41 6.3660.39 6.4860.81 6.6460.Day 4 4.9860.33 4.3660.29 5.3260.17 5.1760.25 4.8060.33 5.0360.26 5.2760.45 5.3360.22 5.1360.13 5.5760.Day 8 4.9260.17 4.5760.12 5.3660.36 5.4160.30 4.9760.42 4.5260.37 5.6460.32 5.9060.21 5.7660.49 5.9160.Day 11 5.4160.17 4.6260.26*(P) 4.3760.24* 4.1760.22** 5.0360.20 4.6160.23*(P) 4.7160.16*(P) 4.6560.12*(P) 4.8260.23 5.1060.Results are shown as mean 6 SEM; Control: 0.25 of methylcellulose, MA: Morus alba leaf extract, 1: chlorogenic acid, 2: rutin, 3: isoquercitrin; * and **: p,0.05 and 0.01, respectively by one-way ANOVA followed by Dunnett’s multiple comparison test, *(P): p,0.05 by one-way ANOVA followed by Bonferroni post test with uncorrected P value and confidence interval, as compared to the control group. doi:10.1371/journal.pone.0050619.tFigure 4. Plasma glucose levels after 11 days of treatment, where significant differences to the control group were found. Results are shown as mean 6 SEM, G: glibenclamide; for further details see Table 1 legend. doi:10.1371/journal.pone.0050619.gAntidiabetic Effect of Major Mulberry Constituentsobserved for chlorogenic acid and rutin but not for isoquercitrin. The two previous compounds were found to play an important role in the anti-diabetic effect of mulberry leaves: ca. half of the observed activity can apparently be 18325633 explained by their presence. Although testing the three compounds was also attempted in combination, at this time no conclusion on the presence or absence of synergistic effect can be made. 4. Based on the above, our analytical method can provide a valuable tool and a reasonable alternative of the existing methods for the quality control of mulberry products.Materials and Methods Ethics statementThe animals were treated in accordance with the European Communities Council Directives (86/609/ECC). The experimental animal protocol satisfied the Guidelines for Animal Experimentation approved by the Animal Experimentation Committee of the University of Szeged (approval no: IV/01758?/2008). Rats were kept at 22 3uC; the relative humidity was 30?0 and maintained on a 12 h light:12 h darkness cycle. The animals were maintained on a standard rodent pellet diet (Charles-River Laboratories, Isaszeg, Hungary) with tap water available ad Asiaticoside A biological activity libitum. After the experiments, they were sacrificed by CO2 inhalation. Field activity for collecting plant sample di.Glucose levels in the different treatment groups.glucose absorption. This would also mean, that in case of a welldesigned mulberry preparation for anti-diabetic purposes the high chlorogenic acid and rutin content should be accompanied by low levels of certain undesired flavonoid(s) ?future research is needed to clarify whether such criteria are necessary or not.ConclusionsOur results can briefly be summarized as follows. 1. A significant, dose-dependent anti-diabetic activity was found for the 70 aqueous ethanolic extract of Morus alba leaves on our in vivo model of type II. diabetic rats. 2. An analitical method was developed for the rapid, selective determination of three, potentially active, major constituents (chlorogenic acid, rutin and isoquercitrin) of the extract by HPLC-DAD. 3. Contribution of the three major constituents to the overall activity was investigated, and a dose related activity wasGroups Control Glibenclamide MA (250 mg/kg) MA (750 mg/kg) 1 (9 mg/kg) 1 (27 mg/kg) 2 (5 mg/kg) 2 (15 mg/kg) 3 (3 mg/kg) 3 (9 mg/kg)Day 0 6.3260.41 5.5260.39 7.3160.80 5.6060.27 6.0860.48 6.1160.41 6.7961.41 6.3660.39 6.4860.81 6.6460.Day 4 4.9860.33 4.3660.29 5.3260.17 5.1760.25 4.8060.33 5.0360.26 5.2760.45 5.3360.22 5.1360.13 5.5760.Day 8 4.9260.17 4.5760.12 5.3660.36 5.4160.30 4.9760.42 4.5260.37 5.6460.32 5.9060.21 5.7660.49 5.9160.Day 11 5.4160.17 4.6260.26*(P) 4.3760.24* 4.1760.22** 5.0360.20 4.6160.23*(P) 4.7160.16*(P) 4.6560.12*(P) 4.8260.23 5.1060.Results are shown as mean 6 SEM; Control: 0.25 of methylcellulose, MA: Morus alba leaf extract, 1: chlorogenic acid, 2: rutin, 3: isoquercitrin; * and **: p,0.05 and 0.01, respectively by one-way ANOVA followed by Dunnett’s multiple comparison test, *(P): p,0.05 by one-way ANOVA followed by Bonferroni post test with uncorrected P value and confidence interval, as compared to the control group. doi:10.1371/journal.pone.0050619.tFigure 4. Plasma glucose levels after 11 days of treatment, where significant differences to the control group were found. Results are shown as mean 6 SEM, G: glibenclamide; for further details see Table 1 legend. doi:10.1371/journal.pone.0050619.gAntidiabetic Effect of Major Mulberry Constituentsobserved for chlorogenic acid and rutin but not for isoquercitrin. The two previous compounds were found to play an important role in the anti-diabetic effect of mulberry leaves: ca. half of the observed activity can apparently be 18325633 explained by their presence. Although testing the three compounds was also attempted in combination, at this time no conclusion on the presence or absence of synergistic effect can be made. 4. Based on the above, our analytical method can provide a valuable tool and a reasonable alternative of the existing methods for the quality control of mulberry products.Materials and Methods Ethics statementThe animals were treated in accordance with the European Communities Council Directives (86/609/ECC). The experimental animal protocol satisfied the Guidelines for Animal Experimentation approved by the Animal Experimentation Committee of the University of Szeged (approval no: IV/01758?/2008). Rats were kept at 22 3uC; the relative humidity was 30?0 and maintained on a 12 h light:12 h darkness cycle. The animals were maintained on a standard rodent pellet diet (Charles-River Laboratories, Isaszeg, Hungary) with tap water available ad libitum. After the experiments, they were sacrificed by CO2 inhalation. Field activity for collecting plant sample di.
